Separation-of-function mutants reveal the NF-κB-independent involvement of IκBα in the regulation of stem cell and oncogenic programs

Abstract

We previously demonstrated that the NF-κB inhibitor IκBα binds the chromatin together with PRC2 to regulate a subset of developmental- and stem cell-related genes. This alternative function has been elusive in both physiological and disease conditions because of the predominant role of IκBα as a negative regulator of NF-κB. We here uniquely characterize specific residues of IκBα that allow the generation of separation-of-function (SOF) mutants that are defective for either NF-κB-related (SOFΔNF-κB) or chromatin-related (SOFΔH2A,H4) activities. Expression of IκBα SOFΔNF-κB, but not SOFΔH2A/H4, is sufficient to negatively regulate a specific stemness program in intestinal cells, thus rescuing the differentiation blockage imposed by IκBα deficiency. In contrast, full IκBα activity is required for regulating clonogenic/tumor-initiating activity of colorectal cancer cells. Our data indicate that SOF mutants represent an exclusive tool for studying IκBα functions in physiology and disease, and identified IκBα as a robust prognosis biomarker for human cancer.

Chromatin enhancer activity of IκBα mediates the exit from naïve pluripotency

“…Similarly, we found that the same IκBα ΔNF-κB mutant is able to restore differentiation capacity in intestinal cells (Álvarez-Villanueva et al 2023). The role of NF-κB in mESC differentiation and pluripotency has been previously explored, and while several reports suggest that NF-κB activity correlates with differentiation and loss of pluripotency (Torres and Watt 2008), others claim that NF-κB inhibition correlates with a decrease in pluripotency gene expression (Armstrong et al 2006).…”

“…Disentangling the chromatin-related function of IκBα from its canonical role as an inhibitor of NF-κB has been challenging, making it difficult to determine its truly biological significance. To tackle this challenge, we engineered separation-of-function (SOF) mutants of IκBα, as detailed in 44 , by identifying specific residues that are essential for binding either NF-κB elements or histones. Interestingly, we have shown that only the histone-binding proficient IκBα mutant (IκBα ΔNF-κB ), which lacks the ability to bind to NF-κB, has the capability to reverse the stabilization of the ground state observed in IκBα-KO mESCs.…”

“…Differentiating this chromatin function of IκBα from its NF-κB inhibitory function has been challenging, making it difficult to determine its significance. To address this, we have engineered separation-of-function (SOF) mutants of IκBα (Álvarez-Villanueva et al 2023) by identifying specific residues that are essential to bind to NF-κB elements or histones. By reintroducing these mutants into IκBαdeficient mESCs, we have shown that only the histone-binding proficient IκBα mutant (IκBα ΔNF-κB ) with deficient NF-κB binding can restore the differentiation capacity of mESCs.…”

“…Uncovering the actual impact of chromatin-related IκBα functions is challenging due to the predominant role of this protein on NF-κB regulation. Recent work from our group has identified the residues in the IκBα protein which are responsible to specifically interact with NF-κB subunits or with histones, and we have generated separation-of-function (SOF) IκBα mutants which retain either NF-κB or chromatin-dependent functions (Álvarez-Villanueva et al 2023). Taking advantage of IκBα -/-mESCs, we generated different doxycycline-inducible IκBα (i-IκBα) expressing cell lines containing either a wildtype IκBα protein (i-IκBα WT ) or one of the SOF IκBα mutants: the one that interacts with NF-κB and it is deficient for H2A/H4 binding (IκBα ΔH2A/H4 ) or the one that interacts with H2A/H4 but not with NF-κB (IκBα ΔNF-κB ) (Figures 4A, S5A).…”

“…Unexpectedly, and in contrast with the pro-differentiation effects of NF-κB activation, depletion of the NF-κB inhibitor IκBα precludes the exit of mESCs from naïve pluripotency associated with an epigenetic rewiring. By using the recently described Separation-Of-Function (SOF) IκBα mutants 32 , we have now demonstrated that pluripotency regulation by IκBα is mainly exerted through its chromatin-related function but independent on its capacity to negatively regulate NF-κB.…”